Pathogenic diversification of the gut commensal Providencia alcalifaciens via acquisition of a second type III secretion system.

Providencia alcalifaciens is a Gram-negative bacterium found in a wide variety of water and land environments and organisms. It has been isolated as part of the gut microbiome of animals and insects, as well as from stool samples of patients with diarrhea. Specific P. alcalifaciens strains encode gene homologs of virulence factors found in other pathogenic members of the same Enterobacterales order, such as Salmonella enterica serovar Typhimurium and Shigella flexneri. Whether these genes are also pathogenic determinants in P. alcalifaciens is not known. Here we have used P. alcalifaciens 205/92, a clinical isolate, with in vitro and in vivo infection models to investigate P. alcalifaciens -host interactions at the cellular level. Our particular focus was the role of two type III secretion systems (T3SS) belonging to the Inv-Mxi/Spa family. T3SS 1b is widespread in Providencia spp. and encoded on the chromosome. T3SS 1a is encoded on a large plasmid that is present in a subset of P. alcalifaciens strains, which are primarily isolates from diarrheal patients. Using a combination of electron and fluorescence microscopy and gentamicin protection assays we show that P. alcalifaciens 205/92 is internalized into eukaryotic cells, rapidly lyses its internalization vacuole and proliferates in the cytosol. This triggers caspase-4 dependent inflammasome responses in gut epithelial cells. The requirement for the T3SS 1a in entry, vacuole lysis and cytosolic proliferation is host-cell type specific, playing a more prominent role in human intestinal epithelial cells as compared to macrophages. In a bovine ligated intestinal loop model, P. alcalifaciens colonizes the intestinal mucosa, inducing mild epithelial damage with negligible fluid accumulation. No overt role for T3SS 1a or T3SS 1b was seen in the calf infection model. However, T3SS 1b was required for the rapid killing of Drosophila melanogaster . We propose that the acquisition of two T3SS by horizontal gene transfer has allowed P. alcalifaciens to diversify its host range, from a highly virulent pathogen of insects to an opportunistic gastrointestinal pathogen of animals.

Neuroendocrine correlates of juvenile amphibian behaviors across a latitudinal cline.

We assessed the macrogeographic and neuroendocrine correlates of behavioral variation exhibited by juveniles, an important life stage for dispersal, across the expansive range of the wood frog. By rearing animals from eggs in a common garden then using a novel environment test, we uniquely demonstrated differential expression of juvenile behaviors among 16 populations spanning 8° latitude. On the individual level, cluster analysis indicated three major behavior profiles and principal component analysis resolved four unique axes of behavior, including escape, foraging, food intake, feeding efficiency. We found that increased escape behavior was associated with lower adrenocorticotropic hormone (ACTH)-induced circulating corticosterone (CORT) levels, however, foraging and food intake behaviors were not associated with either resting or ACTH-induced CORT. At the population level, the expression of foraging behaviors increased with latitude while food intake behaviors declined with latitude, which raised several hypotheses of eco-evolutionary processes likely driving this variation. Given that these behaviors covary along the same ecological gradient as locally adapted developmental traits, genomic studies in this species could provide deep insights into how HPA/I activity is associated with the eco-evolutionary processes that structure intraspecific variation in morphology and behavior.

Intracellular niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes diarrheal disease in humans and animals. During salmonellosis, S. Typhimurium colonizes epithelial cells lining the gastrointestinal tract. S. Typhimurium has an unusual lifestyle in epithelial cells that begins within an endocytic-derived Salmonella-containing vacuole (SCV), followed by escape into the cytosol, epithelial cell lysis and bacterial release. The cytosol is a more permissive environment than the SCV and supports rapid bacterial growth. The physicochemical conditions encountered by S. Typhimurium within the epithelial cytosol, and the bacterial genes required for cytosolic colonization, remain largely unknown. Here we have exploited the parallel colonization strategies of S. Typhimurium in epithelial cells to decipher the two niche-specific bacterial virulence programs. By combining a population-based RNA-seq approach with single-cell microscopic analysis, we identified bacterial genes with cytosol-induced or vacuole-induced expression signatures. Using these genes as environmental biosensors, we defined that Salmonella is exposed to oxidative stress and iron and manganese deprivation in the cytosol and zinc and magnesium deprivation in the SCV. Furthermore, iron availability was critical for optimal S. Typhimurium replication in the cytosol, as well as entC, fepB, soxS, mntH and sitA. Virulence genes that are typically associated with extracellular bacteria, namely Salmonella pathogenicity island 1 (SPI1) and SPI4, showed increased expression in the cytosol compared to vacuole. Our study reveals that the cytosolic and vacuolar S. Typhimurium virulence gene programs are unique to, and tailored for, residence within distinct intracellular compartments. This archetypical vacuole-adapted pathogen therefore requires extensive transcriptional reprogramming to successfully colonize the mammalian cytosol.

Ranavirus infection dynamics and shedding in American bullfrogs: consequences for spread and detection in trade.

American bullfrogs Lithobates catesbeianus are thought to be important in the global spread of ranaviruses-often lethal viruses of cold-blooded vertebrates-because they are commonly farmed, dominate international trade, and may be 'carriers' of ranavirus infections. However, whether American bullfrogs are easily infected and maintain long-lasting ranavirus infections, or are refractory to or rapidly clear infections, remains unknown. We tracked the dynamics of ranavirus in American bullfrogs through time and with temperature in multiple types of samples and also screened shipments from commercial suppliers to determine whether we could detect subclinical infections. Collectively, we found that tadpoles and juveniles were commonly infected at moderate doses, and while some died, others controlled and appeared to clear their infections. Some individuals, however, harbored subclinical infections for up to 49 d, suggesting that American bullfrogs may be important carriers. Indeed, tadpoles and metamorphosed frogs from 2 of 5 commercial suppliers harbored subclinicial infections. Juveniles at warmer temperatures had less intense but still persistent infections. Because diagnostic performance was strongly related to infection intensity, non-lethal samples (i.e. tail or toe clips, swabs, and environmental DNA) had only a moderate chance of detecting subclinical infections. Even internal tissues may fail to detect subclinical infections. However, viral shedding was correlated with the intensity of infection, so while subclinically infected tadpoles shed virus for 35-49 d, the low levels might lead to little transmission. We suggest that a quantitative focus on virus dynamics within hosts can provide a more nuanced view of ranavirus infections and the risk presented by American bullfrogs in trade.

A Brucella Type IV Effector Targets the COG Tethering Complex to Remodel Host Secretory Traffic and Promote Intracellular Replication.

Many intracellular pathogens exploit host secretory trafficking to support their intracellular cycle, but knowledge of these pathogenic processes is limited. The bacterium Brucella abortus uses a type IV secretion system (VirB T4SS) to generate a replication-permissive Brucella-containing vacuole (rBCV) derived from the host ER, a process that requires host early secretory trafficking. Here we show that the VirB T4SS effector BspB contributes to rBCV biogenesis and Brucella replication by interacting with the conserved oligomeric Golgi (COG) tethering complex, a major coordinator of Golgi vesicular trafficking, thus remodeling Golgi membrane traffic and redirecting Golgi-derived vesicles to the BCV. Altogether, these findings demonstrate that Brucella modulates COG-dependent trafficking via delivery of a T4SS effector to promote rBCV biogenesis and intracellular proliferation, providing mechanistic insight into how bacterial exploitation of host secretory functions promotes pathogenesis.

Postreplication Roles of the Brucella VirB Type IV Secretion System Uncovered via Conditional Expression of the VirB11 ATPase.

Brucella abortus, the bacterial agent of the worldwide zoonosis brucellosis, primarily infects host phagocytes, where it undergoes an intracellular cycle within a dedicated membrane-bound vacuole, the Brucella-containing vacuole (BCV). Initially of endosomal origin (eBCV), BCVs are remodeled into replication-permissive organelles (rBCV) derived from the host endoplasmic reticulum, a process that requires modulation of host secretory functions via delivery of effector proteins by the Brucella VirB type IV secretion system (T4SS). Following replication, rBCVs are converted into autophagic vacuoles (aBCVs) that facilitate bacterial egress and subsequent infections, arguing that the bacterium sequentially manipulates multiple cellular pathways to complete its cycle. The VirB T4SS is essential for rBCV biogenesis, as VirB-deficient mutants are stalled in eBCVs and cannot mediate rBCV biogenesis. This has precluded analysis of whether the VirB apparatus also drives subsequent stages of the Brucella intracellular cycle. To address this issue, we have generated a B. abortus strain in which VirB T4SS function is conditionally controlled via anhydrotetracycline (ATc)-dependent complementation of a deletion of the virB11 gene encoding the VirB11 ATPase. We show in murine bone marrow-derived macrophages (BMMs) that early VirB production is essential for optimal rBCV biogenesis and bacterial replication. Transient expression of virB11 prior to infection was sufficient to mediate normal rBCV biogenesis and bacterial replication but led to T4SS inactivation and decreased aBCV formation and bacterial release, indicating that these postreplication stages are also T4SS dependent. Hence, our findings support the hypothesis of additional, postreplication roles of type IV secretion in the Brucella intracellular cycle. IMPORTANCE: Many intracellular bacterial pathogens encode specialized secretion systems that deliver effector proteins into host cells to mediate the multiple stages of their intracellular cycles. Because these intracellular events occur sequentially, classical genetic approaches cannot address the late roles that these apparatuses play, as secretion-deficient mutants cannot proceed past their initial defect. Here we have designed a functionally controllable VirB type IV secretion system (T4SS) in the bacterial pathogen Brucella abortus to decipher its temporal requirements during the bacterium's intracellular cycle in macrophages. By controlling production of the VirB11 ATPase, which energizes the T4SS, we show not only that this apparatus is required early to generate the Brucella replicative organelle but also that it contributes to completion of the bacterium's cycle and bacterial egress. Our findings expand upon the pathogenic functions of the Brucella VirB T4SS and illustrate targeting of secretion ATPases as a useful strategy to manipulate the activity of bacterial secretion systems.

Neutrophils are resistant to Yersinia YopJ/P-induced apoptosis and are protected from ROS-mediated cell death by the type III secretion system.

BACKGROUND: The human innate immune system relies on the coordinated activity of macrophages and polymorphonuclear leukocytes (neutrophils or PMNs) for defense against bacterial pathogens. Yersinia spp. subvert the innate immune response to cause disease in humans. In particular, the Yersinia outer protein YopJ (Y. pestis and Y. pseudotuberculosis) and YopP (Y. enterocolitica) rapidly induce apoptosis in murine macrophages and dendritic cells. However, the effects of Yersinia Yop J/P on neutrophil fate are not clearly defined. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we utilized wild-type and mutant strains of Yersinia to test the contribution of YopJ and YopP on induction of apoptosis in human monocyte-derived macrophages (HMDM) and neutrophils. Whereas YopJ and YopP similarly induced apoptosis in HMDMs, interaction of human neutrophils with virulence plasmid-containing Yersinia did not result in PMN caspase activation, release of LDH, or loss of membrane integrity greater than PMN controls. In contrast, interaction of human PMNs with the virulence plasmid-deficient Y. pestis strain KIM6 resulted in increased surface exposure of phosphatidylserine (PS) and cell death. PMN reactive oxygen species (ROS) production was inhibited in a virulence plasmid-dependent but YopJ/YopP-independent manner. Following phagocytic interaction with Y. pestis strain KIM6, inhibition of PMN ROS production with diphenyleneiodonium chloride resulted in a reduction of PMN cell death similar to that induced by the virulence plasmid-containing strain Y. pestis KIM5. CONCLUSIONS: Our findings showed that Yersinia YopJ and/or YopP did not induce pronounced apoptosis in human neutrophils. Furthermore, robust PMN ROS production in response to virulence plasmid-deficient Yersinia was associated with increased PMN cell death, suggesting that Yersinia inhibition of PMN ROS production plays a role in evasion of the human innate immune response in part by limiting PMN apoptosis.

Yersinia pestis type III secretion system-dependent inhibition of human polymorphonuclear leukocyte function.

Human polymorphonuclear leukocytes (PMNs, or neutrophils) are the primary innate host defense against invading bacterial pathogens. Neutrophils are rapidly recruited to sites of infection and ingest microorganisms through a process known as phagocytosis. Following phagocytosis by human PMNs, microorganisms are killed by reactive oxygen species (ROS) and microbicidal products contained within granules. Yersinia pestis, the causative agent of plague, is capable of rapid replication and dissemination from sites of infection in the host. Although Y. pestis survives in macrophages, the bacterial fate following interaction with human PMNs is less clear. The ability of Y. pestis to inhibit phagocytosis by human PMNs was assessed by differential fluorescence microscopy and was shown to be dependent on expression of the type III secretion system (TTSS). Previous studies have demonstrated that TTSS expression in enteropathogenic Yersinia spp. also inhibits the respiratory burst in PMNs and macrophages, and we show here that human PMN ROS production is similarly repressed by Y. pestis. However, exclusion of uningested TTSS-expressing Y. pestis with gentamicin revealed that intracellular bacteria are eliminated by human PMNs, similar to bacteria lacking the TTSS. In summary, our results suggest that the Y. pestis TTSS contributes to extracellular survival following interactions with human PMNs and that the intracellular fate is independent of TTSS inhibition of neutrophil ROS production.

Aryl hydrocarbon receptor activation impairs the priming but not the recall of influenza virus-specific CD8+ T cells in the lung.

The response of CD8+ T cells to influenza virus is very sensitive to modulation by aryl hydrocarbon receptor (AhR) agonists; however, the mechanism underlying AhR-mediated alterations in CD8+ T cell function remains unclear. Moreover, very little is known regarding how AhR activation affects anamnestic CD8+ T cell responses. In this study, we analyzed how AhR activation by the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the in vivo distribution and frequency of CD8+ T cells specific for three different influenza A virus epitopes during and after the resolution of a primary infection. We then determined the effects of TCDD on the expansion of virus-specific memory CD8+ T cells during recall challenge. Adoptive transfer of AhR-null CD8+ T cells into congenic AhR(+/+) recipients, and the generation of CD45.2AhR(-/-)-->CD45.1AhR(+/+) chimeric mice demonstrate that AhR-regulated events within hemopoietic cells, but not directly within CD8+ T cells, underlie suppressed expansion of virus-specific CD8+ T cells during primary infection. Using a dual-adoptive transfer approach, we directly compared the responsiveness of virus-specific memory CD8+ T cells created in the presence or absence of TCDD, which revealed that despite profound suppression of the primary response to influenza virus, the recall response of virus-specific CD8+ T cells that form in the presence of TCDD is only mildly impaired. Thus, the delayed kinetics of the recall response in TCDD-treated mice reflects the fact that there are fewer memory cells at the time of reinfection rather than an inherent defect in the responsive capacity of virus-specific memory CD8+ cells.

A dose-response study of the effects of prenatal and lactational exposure to TCDD on the immune response to influenza a virus.

The goal of the current study was to evaluate the immune response to a common respiratory pathogen, influenza A virus, in mice exposed to increasing doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during development. Additionally, the treatment paradigm was designed to provide exposure throughout fetal and neonatal development, beginning on d 1 of gestation. To accomplish this, impregnated C57Bl/6 mice were treated with 0.25 microg/kg TCDD on d 0 and 7 of pregnancy, followed by 2 additional doses of 0.25, 1, or 5 microg/kg given on d 14 and postpartum d 2. The adult offspring were infected with influenza virus, and components of the adaptive and innate immune responses were evaluated. Our results show that developmental exposure to TCDD dose-responsively suppressed both the cell-mediated and antibody responses to influenza virus in female but not males. In contrast, TCDD exposure enhanced the innate immune responses in offspring of both sexes; specifically, neutrophilia and interferon (IFN) gamma levels in the lung were increased. These alterations in functional immunity did not result from overt toxicity to the immune organs, as developmental TCDD exposure did not alter the cellular composition of the thymus, spleen, or bone marrow. These findings extend our knowledge of the dose-responsive nature of immunological defects induced by developmental exposure to TCDD and offer insight regarding the dose required to alter the immune response to viral infection. Moreover, we demonstrate a clear dose at which no observable effects on immune function later in life were detected.

A novel effect of dioxin: exposure during pregnancy severely impairs mammary gland differentiation.

Many ligands for the aryl hydrocarbon receptor (AhR) are considered endocrine disruptors and carcinogens, and assessment of adverse health effects in humans exposed to such chemicals has often focused on malignancies, including breast cancer. Mammary tissue contains the AhR, and inappropriate activation of the AhR during fetal development causes defects in mammary development that persist into adulthood. However, it is not known whether the extensive differentiation of mammary tissue that occurs during pregnancy is also sensitive to disruption by AhR activation. To examine this, we exposed pregnant C57Bl/6 mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on days 0, 7, and 14 of pregnancy. Examination of mammary glands on days 9, 12, and 17 of pregnancy and on the day of parturition showed severe defects in development, including stunted growth, decreased branching, and poor formation of lobular alveolar structures. This impaired differentiation was biologically significant, as expression of whey acidic protein in the gland was suppressed, and all pups born to TCDD-treated dams died within 24 h of birth. Analysis of circulating progesterone, prolactin, and estradiol suggest that hormone production was slightly impaired by inappropriate activation of the AhR. However, hormone levels were affected only very late in pregnancy. Given that the observed defects in gland development preceded these hormonal effects, altered hormone levels are an unlikely mechanistic explanation for impaired mammary development. This novel finding that AhR activation during pregnancy disrupts mammary gland differentiation raises questions about the susceptibility of mammary tissue to direct injury by endocrine disrupting agents and the potential for AhR-mediated signaling to adversely affect lactation and breast tissue development in human populations.

Developmental exposure to the potent aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin Impairs the cell-mediated immune response to infection with influenza a virus, but enhances elements of innate immunity.

Based on demonstrated effects on functional immunity in rodent models and supportive evidence from epidemiological studies, it is apparent that developmental exposure to ligands for the aryl hydrocarbon receptor (AhR) has the potential to impair immunity in human populations. Furthermore, due to the high levels of these compounds detected in human breast milk, and the fact that they cross the placenta, it is clear that humans are exposed to AhR ligands during fetal and neonatal development. The current studies were conducted to further characterize the relationship between developmental exposure to TCDD, the most potent AhR agonist, and defects in immune function later in life. Impregnated C57Bl/6 mice were treated with 4 doses of 1 mircog/kg TCDD, given on days 0, 7, and 14 of pregnancy, and 2 days after parturition. Functional immunity was assessed by challenging the adult offspring with influenza virus. Both male and female offspring of the TCDD-treated dams demonstrated impairment of the adaptive immune response, as evidenced by suppressed numbers of T cells and IFNgamma-producing cells in the draining lymph nodes and reduced T cell recruitment to the lung. In contrast, the inflammatory response, including infection-associated pulmonary neutrophilia and IFNgamma levels, was significantly elevated in the developmentally-exposed mice. These functional defects in immunity were not correlated with defects in hematopoeisis, as immune cells in the bone marrow, spleen, and thymus were phenotypically normal in uninfected mice. These results support the idea that immune alterations that arise during development cause persistent and significant changes in immune function.